In the specification, reference is made to a number of documents from the prior art including patent applications and manufacturer's manuals. While the disclosure of these documents is not considered relevant to the patentability of the invention, it is incorporated by reference into the present specification.
Dendritic cells (DCs) are the link between the innate and the adaptive immune response. They are able to detect pathogens as such, and to initialize and direct an adaptive (that is, tailored to the specific pathogen) immune response. In the absence of pathogens, DCs are also capable of mediating tolerance against endogenous antigens. Thus, DCs are the key for the targeted induction of immune responses, but also for mediating immunological tolerance. The cytokine IL-12p70 plays an important role in the induction of cell mediated immunity, while the cytokine IL-10 is involved in the induction of humoral immunity, and also of tolerance. Immunotherapy of malignant diseases using DCs as adjuvants was tested already in various clinical trials, where the safety and feasibility of this method could be demonstrated. However, the clinical outcomes remained below expectations, although frequently the patients' immune responses against the antigens used were detectable in vitro. Generating the DCs in cell culture offers the opportunity to manipulate them in a targeted way. For this purpose, it is advantageous to generate DCs, which are able to induce long-lived memory T cells, and thereby act resistant to regulatory T cells (Tregs) and other tolerogenic mechanisms. Upon re-exposure to an antigen, long-lived memory T cells can mediate a more rapid and more efficient secondary response. This memory function can be provided by CD4+ and CD8+ memory T cells. Long-lived memory T cells are different from effector cells that only have a short life time and usually die after an immune response by activation-inducing cell death (AICD). Between the two cell types, however, there are transitional forms, such as the effector memory cells. Like effector cells, they are able to patrol throughout the body, and exert an effector function upon antigen contact, and they can proliferate and are also more long-lived than effector cells. On the other hand, the use of DCs for the targeted treatment of autoimmunity and allergies is conceivable because DCs can suppress immune responses and mediate tolerance under certain conditions. Improved methods and protocols for the manufacture of various types of DCs are therefore of great interest and are the object of research worldwide. An overview of the current prior art is presented in Boczkowski and Nair, Expert Opin. Biol. Ther. 10 (4) (2010), 563-574, and in Kaisho and Tanaka, Trends in Immunology 29 (7) (2008), 329-336.
DCs have a variety of surface receptors with which they can identify various pathogens. In addition, DCs are able to perceive various endogenous messengers such as cytokines and chemokines, and surface molecules on other cells of the immune system. The DC processes the various incoming signals via intracellular signaling pathways, whereby various differentiation programs are triggered. The targeted manipulation of these signaling pathways may allow the creation of tailor-made DCs, which are thus better suited to mediate either immunity (in cancer immunotherapy) or tolerance (in the treatment of autoimmune diseases and allergies).
Different ways to intervene in the signaling pathways in the DC through genetic manipulation have already been formulated and implemented. However, various barriers get in the way, especially in the manipulation of human DCs. Genetic manipulation in the context of a therapy gives cause for concern, and somatic gene therapy is tightly regulated. In addition, the options of genetically modifying the human DCs most widely used in medicine (monocyte-derived DCs) are very limited, and only the use of viral transfection systems, which were developed from lentiviruses or adenoviruses, has been successful so far. The use of such vehicles for introducing DNA, however, has been viewed very critically, and bears additional risks. For example, by using lentiviruses, viral sequences are also always incorporated into the genome of the cell. This may destroy active endogenous genes, or the viral promoters may activate genes that would otherwise be inactive. Since integration into the genome is random, however, it is impossible to predict which genes may be affected. If tumor suppressor genes or oncogenes are destroyed or activated, the cell, in the worst case, may become a tumor cell. Also, the induced immune response may be directed against the viral products rather than against the desired antigens. The latter also applies to adenoviral systems, where, in this case, the immune response may be very severe, since many people already have an existing immune response against adenoviruses. In 1999, such a severe immune response against an adenoviral vector even resulted in a fatality.
A central signaling pathway of the DC is the NFκB signaling cascade. Stimulation of many of the surface receptors of the DC leads to activation of this cascade, wherein inhibitory proteins are destabilized by phosphorylation, so that transcription factors will enter the nucleus where they cause the transcription of various genes. The kinases that perform such phosphorylation are called IKK (inhibitor of kappa kinases).